Dosing-time dependent effect of dexamethasoneon bone density in rats

著者 Takahashi Masaki, Ushijima Kentarou, HayashiYohei, Maekawa Tomohiro, Ando Hitoshi,Tsuruoka Shu-ichi, Fujimura Akio

journal orpublication title

Life sciences

volume 86number 1-2page range 24-29year 2010-01権利 (C) 2009 Elsevier Inc.URL http://hdl.handle.net/2241/104423

doi: 10.1016/j.lfs.2009.10.020

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Title: Dosing-time dependent effect of dexamethasone on bone density in rats

Masaki Takahashi, Kentarou Ushijima, Yohei Hayashi, Tomohiro Maekawa, Hitoshi Ando,

Shu-ichi Tsuruoka, Akio Fujimura

Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical University,

Tochigi, Japan (M.T., K.U., Y.H., T.M., H.A., and A.F.); Department of Nephrology, Institute

of Clinical Sciences, University of Tsukuba, Ibaraki, Japan (S.T)

Address: Division of Clinical Pharmacology, Department of Pharmacology, Jichi Medical

University, Tochigi 329-0498, Japan

Phone: +81-285-58-7388

Fax: +81-285-44-7562

Corresponding Author: Akio Fujimura, MD, Ph.D.

E-mail address: akiofuji@jichi.ac.jp

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Structured abstract

Aims: While glucocorticoids are widely used to treat patients with various diseases, they

often cause adverse effects such as bone fractures. In this study, we investigated whether the

decrease in bone density induced by glucocorticoid therapy was ameliorated by optimizing a

dosing-time.

Main methods: Rats were administered with dexamethasone (Dex) orally (1 mg/kg/day) for

6 weeks at a resting or an active period. After the end of the treatment, bone density of

femur, biomarkers of bone formation and resorption, and other biomedical variables were

measured.

Key findings: Bone density of femur was significantly decreased by the 6-week treatment

with Dex, and the degree of decrease in the 14 HALO (hours after light on) dosing group (an

active period) was larger than that in the 2 HALO dosing group (a resting period). Although

urinary calcium excretion was accelerated by Dex treatment, secondary hyperparathyroidism

was not detected. Histomorphometry analysis showed that Dex suppressed bone resorption,

which was larger in the 2 HALO than in the 14 HALO groups. These data indicate that Dex

equally suppressed bone formation in the 2 and 14 HALO groups, but inhibited bone

resorption more in the 2 HALO than in the 14 HALO groups.

Significance: This study shows that the decrease in bone density induced by Dex was

changed by its dosing-time.

Key words; chronopharmacology, dexamethasone, osteoclasts, osteoporosis, urinary calcium

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Introduction

Glucocorticoids (GCs) are widely used to treat patients with inflammatory disorders such

as obstructive airway disease, rheumatoid arthritis and inflammatory bowel diseases.

Unfortunately GCs cause several kinds of adverse effects involving hyperglycemia,

hypertension and sleep disturbance (Andrews and Walker 1999; Ling et al. 1981; Whitworth

et al. 2000). It is well known that the repeated treatment with GCs also increases the risk of

bone fractures (Kanis et al. 2004; Van Staa et al. 2000a), which is dose-dependent and occurs

rapidly after the initiation of the treatment (Van Staa et al. 2000b). In addition, a recent

database analysis showed that the high-dose of oral GCs leads to the increased risk of

osteoporotic fracture (De Vries et al. 2007).

Bone metabolism is a dynamic and continuous remodeling process that is normally

maintained in a tightly coupled balance between the resorption of old or injured bone and the

formation of new bone (Dempster 1992). In brief, osteoclast precursors are recruited to a

bone surface (activation), and then the newly formed osteoclasts remove both the mineral and

organic components of bone matrix (resorption). Osteoblasts and their precursor assemble to

refill the resorption cavity. Bone formation is beginning with the deposition of osteoid by

the osteoblasts, and then the second stage is mineralization of the organic matrix. Bone

remodeling serve two principal functions as follows: renewing the skeleton continuously, and

playing a role in mineral homeostasis by transferring calcium and other ions.

It is important to increase the therapeutic effects of drugs and to decrease their adverse

effects. Chronotherapy is one of the approaches to achieve these goals by optimizing a

dosing-time. We already demonstrated the merits of chronotherapy using several drugs in

animals and human subjects (Kitoh et al. 2005; Nozawa et al. 2006; Tsuruoka et al. 2004b;

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Ushijima et al. 2005). Dosing-time dependent changes in the pharmacokinetics and/or

pharmacodynamics are mainly involved in chronopharmacological phenomenon of drugs

(Lemmer 2005).

There are many reports indicating that the circadian rhythmicity is a typical feature of bone

metabolism. For example, plasma concentrations of calcium and its regulating hormones

(Fraser et al. 1998; Mühlbauer and Fleisch 1995), and markers of bone formation and

resorption (Greenspan et al. 1997; Nielsen et al. 1991; Shao et al. 2003; Blumsohn et al. 1994;

Bollen et al. 1995) showed circadian rhythms. In addition, the influences of drugs on bone

metabolism are reported to be altered by their dosing-time (Schlemmer et al. 1997; Tsuruoka

et al. 2002, 2004a, 2004b, 2007). Based on these data, we hypothesized that the decrease in

bone density induced by GCs therapy is ameliorated by optimizing a dosing-time.

To address this issue, we examined the influence of GC dosing-time on the decrease in

bone density, and evaluated a potential mechanism involving in this phenomenon. In this

study, we used dexamethasone, which is a potent synthetic member of the glucocorticoid

family.

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Materials and Methods

Animal and chemicals

Six-week old male Wistar rats were obtained from Japan SLC Co. (Shizuoka, Japan).

They were maintained for more than 2 weeks before the experiment in two rooms under a

specific pathogen-free environment and a 12-hour light/dark cycle. The lights were switched

on and off at 07:00 and 19:00 in room 1, and at 19:00 and 07:00 in room 2. Rats had free

access to standard chow and water. Chow diet used in this study (CE-2, Crea Japan, Tokyo,

Japan) contained 1.06% of calcium, 0.98% of phosphorus and 220IU/100mg of Vitamin D.

The experiments were performed in accordance with EC Directive 86/609/ECC and the Use

and Care of Experimental Animals Committee of Jichi Medical University (Tochigi, Japan).

Dexamethasone (Dex) was purchased from Sigma-Aldrich (St Louis, MO), and was

suspended in the distilled water. Calcein was obtained from Wako Pure Chemical Industries

(Osaka, Japan) and dissolved in 2% NaHCO3 solution.

Drug dosing and sample collection

After the acclimatization period, rats (n=24) were divided into four groups, and Dex (1

mg/kg p.o.) or vehicle was given by a gastric gavage at two different times [2 or 14 hour after

lights on (HALO), Fig. 1] once a day for 6 weeks;

Group 1: vehicle at 2 HALO (n=6), Group 2: Dex at 2 HALO (n=6),

Group 3: vehicle at 14 HALO (n=6), Group4: Dex at 14 HALO (n=6).

At the final day, rat was administered with 3% of body weight of deionized water at 30 min

after dosing and separately placed in a metabolic cage for 4 hours to collect urine sample.

Twenty four hours after the last dosing, rat was anesthetized with pentobarbital sodium (50

mg/kg i.p.), and femur and blood were collected. Femur was stored in 70% ethanol, and

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serum samples were stored at -80 oC until assay.

For histomorphometry analysis, rats (n=12) were divided into four groups and received the

dosing of Dex or vehicle for 6 weeks as described above. Animal was injected with calcein

(8 mg/kg i.p.) at 10 and 3 days before the last dosing. Twenty four hours after the last

dosing, femur was obtained.

Measurement of femur bone density

The bone density of femur was determined by dual energy X-ray absorption (DCS-600A,

Aloka, Japan). The scan was performed every 2 mm along the axis of the bone from

proximal end, and 18 scans were obtained for each bone. Average of the first proximal 3

scans, middle part of 4 scans, and the last part of 3 scans were regarded as “proximal”,

“medial” and “distal”. Average of all scans in bone was shown as “whole”. “Medial” is

exclusively cortical bone and “distal” is rich in cancellous bone (Tsuruoka et al. 2002).

Assays

Serum osteocalcin concentration was determined by ELISA using a commercialized kit

(Biomedical Technologies Inc., Stoughton, MA). Assay was performed according to the

instruction manual. The intra- and inter-assay coefficients of variation were better than 7%.

Urinary C-terminal telopeptides of type I collagen (CTx) concentration was measured by

ELISA using a commercialized kit (RatLaps™ EIA, Immunodiagnostic systems, Tyne and

Wear, UK). Creatinine concentration in urine was measured by an enzyme method (Sekisui

Medical Co., Ltd., Tokyo, Japan) by an autoanalyzer. The value of urinary CTx was corrected

by creatinine concentration.

Calcium concentration in serum and urine was measured by the orthocresolphthalein

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complex method (Mitsubishi Kagaku Iatron, Inc., Tokyo, Japan) with an autoanalyzer (7170,

Hitachi Ltd., Tokyo, Japan).

Serum parathyroid hormone (PTH) concentration was determined by an

immunoradiometric assay (Rat PTH IRMA kit, Immunotopics, Inc., San Clemente, CA). Its

normal range was 10-40 pg/ml in rats (Tsuruoka, et al. 2002).

Bone Histomorphometry

Femur was fixed in 70% ethanol and embedded in glycolmethacrylate without

decalcification, and sectioned in 3-mm slices. The sections were stained with Toluidine blue

not only to discriminate between mineralized and unmineralized bone, but to identify cellular

components. Histomorphometry was performed with semiautomated image analyzing

system (OsteoplanII; Carl Zeiss, Thornwood, NY), which was linked to a light microscope at

200-fold magnification. Parameters using the trabecular bone were measured in an area 2.4

mm in length from 0.6-1.2 mm below the growth plate. The following parameters were

calculated; trabecular bone volume expressed as a percentage of total tissue volume (BV/TV),

percentage of osteoid volume (OV/BS), percentage of osteoid surface (OS/BS), percentage of

osteoblasts surface (Obs/BS), number of mature osteoclasts in 10 cm of bone perimeter

(N.Oc/B.Prr), percentage of bone surface covered by mature osteoclasts (OcS/BS) and

percentage of eroded surface (ES/BS). The thickness of the epiphyseal growth plate

cartilage was measured over a length of approximately 2.5 mm in visual areas. The number

of proliferative cells and hypertrophic cells was counted in the same visual areas of 0.6 mm

inside from epiphyseal side, and were expressed as the average values in six to nine clearly

visible columns.

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Statistical analysis

Data are shown as the means ± S.D. Comparisons between two groups were done by

one-way analysis of variance followed by Bonferroni-Dunn test using StatView (SAS Institute,

Cary, NC). P <0.05 was considered to be significant.

Results

Influence of Dex dosing-time on bone density of femur

Bone densities of femur in the vehicle-treated rats were not significantly different between

the 2 and 14 HALO groups. Repeated treatment with Dex decreased bone density in the 2

and 14 HALO groups (Fig. 2). The parameter in the 14 HALO group was lower than that in

the 2 HALO group, especially in “distal”, “medial” and “whole” sections.

Influence of Dex dosing-time on body weight and food intake

The body weights of Dex-treated groups similarly decreased during the experiment period

while the parameter in the vehicle-treated groups increased (Fig. 3). Food intake did not

significantly differ at any observation points between the vehicle and Dex groups.

Influence of Dex dosing-time on serum osteocalcin concentration

To evaluate the influence of Dex on the bone formation, serum osteocalcin concentration

was measured. Serum osteocalcin concentration was significantly decreased by Dex

treatment in the 2 and 14 HALO groups (Fig. 4). There was no significant difference in this

parameter between the two groups.

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Influence of Dex dosing-time on urinary calcium excretion, and serum concentrations of

PTH and calcium

The ratio of u-calcium/u-creatinine was significantly increased in the Dex dosing group at

14 HALO, but not at 2 HALO (Fig. 5a). Serum PTH concentrations in the Dex-treated

groups elevated, but it did not reach to a statistical significance (Fig. 5b). However, serum

calcium concentrations in the Dex-treated groups were significantly higher than those in the

vehicle groups (Fig. 5c).

Influence of Dex dosing-time on urinary CTx concentration

To evaluate the influence of Dex on bone resorption, urinary CTx concentration was

measured. Urinary CTx concentration tended to be decreased by Dex treatment in the 2 and

14 HALO groups (Fig. 6).

Influence of Dex dosing-time on histomorphometry of trabecular bones of femur

Histomorphometory data did not significantly differ between the vehicle-treated 2 and 14

HALO groups (Table 1a). Bone volume (BV/TV) of the Dex-treated group at 14 HALO was

significant lower than that in the vehicle group, while the parameter in the 2 HALO group did

not significantly decrease. The percentage of osteoid volume (OV/BS), percentage of

osteoid surface (OS/BS), percentage of osteoblasts surface (Obs/BS) and the numbers of

mature osteoclasts (N.Oc/B.Prr) were significantly decreased by Dex treatment in both groups.

However, bone surface covered by mature osteoclasts (OcS/BS) and consequent eroded

surface (ES/BS) were significantly reduced by the treatment with Dex at 2 HALO, but not at

14 HALO.

Histomorphometory data of the epiphyseal growth plate cartilage were shown in Table 1b.

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The thickness of growth plate cartilage and the number of proliferative cells per cell column

were significantly decreased by the Dex- treated 2 and 14 HALO groups. There were no

significant differences in these parameters between the 2 and 14 HALO groups with Dex.

Discussion

As a preliminary study, we tested the 0.1, 0.32 and 1 mg/kg of Dex administration for 6

weeks at 2 HALO, and found that only 1 mg/kg of Dex caused a significant decrease in bone

density of femur (data not shown). Therefore, we selected the 1 mg/kg of Dex

administration for 6 weeks in this study. The preliminary study also showed that body

weights were decreased by 0.32 and 1 mg/kg of Dex in rats. These data suggest that the

kinds of Dex-induced adverse effects depend on its dose. Although body weights were

decreased by Dex treatment at 2 and 14 HALO in this study, food intake did not be decreased

by Dex treatment at any observation points, which indicates that Dex-induced bone loss might

not be due to a reduced nutrition.

In this study, Dex treatment for 6 weeks reduced bone density of femur in rats. Similar

findings were reported as follows; 1) Methylprednisolone reduced the bone mechanical

strength due to the decreased bone quantity and quality (Ortoft and Oxlund 1988). 2)

Repeated treatment with prednisolone caused osteopenia (Lindgren et al. 1983; Goulding and

Gold 1988). However, King et al reported the opposite observation that Dex treatment

increased the trabecular bone volume both in intact and parathyroidectomized rats (King et al.

1996). In their study, Dex was given continuously by an osmotic pomp, and a dose of Dex

(16.25 µg/rat/day) was small. Although we did not have any definite explanations for the

discrepancy of the influence of GCs on bone tissue, it may reside in the differences in the

dose of GC and its dosing route.

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GCs influence the bone metabolism through the multiple pathways as follows; 1) Direct

inhibition on the proliferation of osteoblast (Canalis 1996; Leclerc et al. 2005), 2)

Hyperparathyroidism induced by the direct effect on parathyroid gland (Zhang et al. 1993) or

the GCs-related increase in urinary calcium excretion (Canalis 1996; Reid 1997), and 3)

Direct stimulation (Kaji etb al. 1997; Takuma et al. 2003) or inhibition (Dempster et al. 1997;

Kim et al. 2006) on the formation of osteoclast.

Dex-induced decrease in bone density in the 14 HALO group was larger than that in the 2

HALO group in this study. To evaluate the mechanism(s) involving in the dosing-time

dependent change in the effect of Dex on bone density, we examined the influence of its

dosing-time on bone formation. GCs directly impair the proliferation of osteoblasts and

subsequently reduce the number and function of mature osteoblasts leading to a decrease in

osteocalcin transcription (Canalis 1996; Leclerc et al. 2005). In this study, we measured

serum osteocalcin concentration, a biomarker of osteoblasts after 6-week of dosing. The

parameter was significantly decreased by the treatment with Dex, but there was no significant

difference between the 2 and 14 HALO groups. Therefore, a dosing-time dependent change

in the effect of Dex on bone formation might not be involved in the mechanism of this

phenomenon.

GCs increase urinary calcium excretion by the direct effect on the kidney, which leads to

secondary hyperparathyroidism (Canalis 1996; Reid 1997). Hyperparathyroidism, in turn,

activates osteoclasts, which accelerates calcium release from bone tissue and consequently

causes bone fracture. Thus, the increased urinary calcium excretion is likely a marker of

bone loss induced by Dex. In this study, we determined whether the effect of Dex on the

urinary calcium excretion and serum PTH concentration were influenced by its dosing-time.

The urinary calcium excretion significantly elevated in the Dex-treated 14 HALO group, and

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tended to elevate in the Dex-treated 2 HALO group. These data led us to speculate that Dex

dosing at 14 HALO caused hyperparathyroidism, which resulted in the decrease of bone

density. However, serum PTH concentration did not elevate by Dex treatment and remained

within the normal range between 10-40 pg/ml (Tsuruoka et al. 2002) in the 2 and 14 HALO

groups. Thus the role of PTH on a dosing-time dependent effect of Dex might be small, if

any.

In general, hypercalcemia decreased PTH secretion. In this study, rats treated with Dex

showed hypercalcemia and relatively higher PTH concentraion. It is reported that Dex

accelerated the secretion of PTH from cultured parathyroid cells and elevated PTH mRNA

level (Zhang et al. 1993). Therefore, we think that the direct effect of Dex on parathyroid

gland is reflected in the relatively higher PTH observed in this study.

Since serum calcium concentration was significantly elevated by Dex treatment, it was

speculated that calcium release from bone tissue might be accelerated. To evaluate a

dosing-time dependent effect of Dex on bone resorption, we measured urinary CTx

concentration, a marker of bone resorption, and performed the histomorphometry analysis

using a fluoroscent reagent, calcein. Although all parameters of bone resorption in

histomorphometry analysis were decreased by Dex treatment, the degrees of the reduction

were significantly larger in the 2 HALO than in the 14 HALO groups. The changes in

urinary CTx concentration were comparable with the histomorphometry data. Based on

these data, we think that the activity of bone resorption was greater in the 14 HALO than in

the 2 HALO groups. Histomorphometry analysis showed that the number of osteoblasts was

significantly reduced by Dex in the 2 and 14 HALO groups, which was identical to the result

of serum osteocalcin concentration. In addition, the thickness of epiphyseal growth plate

cartilage and the number of proliferative cells were similarly decreased by Dex in the 2 and

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14 HALO groups. Thus, the Dex-induced suppression on bone formation and epiphyseal

cartilage metabolism did not differ between the 2 and 14 HALO groups. These data indicate

that a dosing-time dependent change in the effect of Dex on bone resorption is involved in

this phenomenon.

The effects of GCs on bone resorption are complex and less clear. Previous in vitro

studies demonstrated that GCs increase the formation and activity of osteoclasts (Kaji et al.

1997; Takuma et al. 2003), while other studies showed that GCs inhibit the osteoclasts

formation by altering the transcription and enhancing apoptosis (Dempster et al. 1997; Kim et

al. 2006). Moreover, a higher dose of methylprednisolone (~ 20 mg/kg) reduced the number

of osteoclasts in rats (Hulley et al. 2002; Wang et al. 2002), but a lower dose of Dex did not

(King et al. 1996). These findings and present data suggest that a higher dose of GCs

inhibits the formation of osteoclasts. However, because pharmacological dose was used in

this study, it is unclear whether chronophrmacological phenomena observed in rats are also

detected in human subjects treated with Dex. Human study is needed to evaluate a potential

dosing-time dependent effect of Dex on bone.

Conclusion

The decrease in bone density induced by Dex was larger in the 14 HALO than in the 2

HALO groups. Dex equally suppressed bone formation in the 2 and 14 HALO groups,

while it inhibited bone resorption more in the 2 HALO than in the 14 HALO groups, which

might cause the dosing-time dependent effect of Dex on bone loss in rats.

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Acknowledgement

We thank Mr. Hisashi Murayama (KUREHA SPECIAL LABORATORY Co., Ltd.) for

technical assistance in bone histomorphometry analysis.

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Table 1a Bone histomorphometory analysis of trabecular bone in femur

2 HALO

14 HALO

vehicle Dex vehicle Dex

BV/TV (%) 19.94 ± 0.51 18.57 ± 1.09 20.48 ± 1.61 16.94 ± 1.06*

OV/BV (%) 0.417± 0.195 0.018± 0.003* 0.307± 0.137 0.017± 0.198*

OS/BS (%) 3.76 ± 1.53 0.14 ± 0.04* 2.78 ± 1.24 0.15 ± 0.05*

Ob.S/BS (%) 4.32 ± 1.88 0.05 ± 0.04* 3.52 ± 1.89 0.19 ± 0.16*

N.Oc/B.Prr (/100mm) 136.78 ± 37.06 42.11 ± 9.68** 133.24 ± 16.54 64.87 ± 6.94*

Oc.S/BS (%) 2.81 ± 0.68 0.51 ± 0.08** 1.94 ± 0.649 0.76 ± 0.14

ES/BS (%) 5.70 ± 0.49 1.57 ± 0.31**, # 4.58 ± 0.85 3.22 ± 0.30

Mean ± S.D. n=3 in each group

*, P <0.05, **, P <0.01 vs. vehicle; #, P <0.05 vs. 14 HALO Dex

- 21 -

Table 1b Histomorphometory analysis of the epiphyseal growth plate cartilage

2 HALO

14 HALO

vehicle Dex vehicle Dex

Thickness (µm) 144.17 ± 15.40 87.15 ± 11.42** 151.41 ± 7.25 95.52 ± 5.11**

No. of proliferative cells per cell column

7.65 ± 0.51 4.04 ± 0.06** 7.91 ± 0.42 3.94 ± 0.85**

No. of hypertrophic cells per cell column

3.32 ± 0.39 2.93 ± 0.48 2.81 ± 0.17 2.11 ± 0.11

Mean ± S.D. n=3 in each group

**, P <0.01 vs. vehicle

- 22 -

Captions

Fig. 1 Schematic representation of two reversed lighting regimens to provide two different

dosing times at one point. By reversing the lighting condition in two different rooms, dosing

at 09:00 approximates the treatment at two circadian stages: 2 and 14 HALO (hour(s) after

lights on).

Fig. 2 Influence of Dex dosing-time on bone density of femur

Dex or vehicle was given once daily at 2 HALO or 14 HALO for 6 weeks. , vehicle at

2HALO; , Dex at 2 HALO; ,vehicle at 14 HALO; , Dex at 14 HALO. Mean ± S.D.,

n=6 in each group *, P <0.05, **, P <0.01 vs. vehicle; #, P <0.05 vs. Dex at 2 HALO

Fig. 3 Influence of Dex dosing-time on body weight and food intake

(a, b); , vehicle; , Dex. (c, d); , vehicle; , Dex. Mean ± S.D., n=6 in each

group **, P <0.01 vs. vehicle

Fig. 4 Influence of Dex dosing-time on serum osteocalcin concentration

Mean ± S.D., n=6 in each group **, P <0.01 vs. vehicle

Fig. 5 Influence of Dex dosing-time on (a) the ratio of urinary calcium/creatinine, (b) serum

PHT and (c) serum calcium concentration

Rats were given with 3% of body weight of deionized water at 30 min after the last dosing,

and placed into a metabolic cage for 4 hr to collect urine. Blood sample was obtained at 24

hr after the last dosing. Mean ± S.D., n=6 in each group *, P <0.05; **, P <0.01 vs. vehicle

- 23 -

Fig. 6 Influence of Dex dosing-time on urinary CTx concentration

Urinary CTx concentration was corrected by creatinine concentration. Mean ± S.D., n=6

in each group

- 24 -

Fig. 1

Room 1

(Standard lighting)

Lights off(active period)

on on

0:00 07:00 19:00 24:00

Lights on(resting period)

off off

Room 2

(Reversed lighting)

2 HALO(09:00)

14 HALO

Room 1

(Standard lighting)

Lights off(active period)

on on

0:00 07:00 19:00 24:00

Lights on(resting period)

off off

Room 2

(Reversed lighting)

2 HALO(09:00)

14 HALO

- 25 -

Fig. 2

80

90

100

110

120

130

140

distal medial proximal whole

***

****

** ***

##

#

Bon

e de

nsity

(mg/

cm2 )

80

90

100

110

120

130

140

distal medial proximal whole

***

****

** ***

##

#

Bon

e de

nsity

(mg/

cm2 )

- 26 -

Fig. 3

0

20

40

60

80

100

0 2 4 6

Food

inta

ke (g

/kg

BW

)

Weeks

(c) 2 HALO

0

20

40

60

80

100

0 2 4 6

Food

inta

ke (g

/kg

BW

)

Weeks

(c) 2 HALO

0

20

40

60

80

100

0 2 4 6

Food

inta

ke (g

/kg

BW

)

Weeks

(d) 14 HALO

0

20

40

60

80

100

0 2 4 6

Food

inta

ke (g

/kg

BW

)

Weeks

(d) 14 HALO

-60

-30

0

30

60

90

120

0 1 2 3 4 5 6

BW

cha

nge

(g)

Weeks

(b) 14 HALO

**

-60

-30

0

30

60

90

120

0 1 2 3 4 5 6

BW

cha

nge

(g)

Weeks

(b) 14 HALO

**

-60

-30

0

30

60

90

120

0 1 2 3 4 5 6

BW

cha

nge

(g)

Weeks

(a) 2 HALO

**

-60

-30

0

30

60

90

120

0 1 2 3 4 5 6

BW

cha

nge

(g)

Weeks

(a) 2 HALO

**

- 27 -

Fig. 4

0

10

20

30

40

50

60

2 HALO

** **

Seru

m o

steo

calc

in(n

g/m

l)

vehicle Dex

14 HALOvehicle Dex

0

10

20

30

40

50

60

2 HALO

** **

Seru

m o

steo

calc

in(n

g/m

l)

vehicle Dex

14 HALOvehicle Dex

- 28 -

Fig. 5

0

2

4

6

8

10

12 **

Seru

m C

a (m

g/dL

)

2 HALOvehicle Dex

14 HALOvehicle Dex

*(c)

0

2

4

6

8

10

12 **

Seru

m C

a (m

g/dL

)

2 HALOvehicle Dex

14 HALOvehicle Dex

*(c)

2 HALOvehicle Dex

14 HALOvehicle Dex

0

10

20

30

40

PTH

(pg/

ml)

(b)

2 HALOvehicle Dex

14 HALOvehicle Dex

0

10

20

30

40

PTH

(pg/

ml)

(b)

0.0

0.2

0.4

0.6

0.8

1.0**

The

ratio

of u

Ca/

uCr(

mM

/nM

)

2 HALOvehicle Dex

14 HALOvehicle Dex

(a)

0.0

0.2

0.4

0.6

0.8

1.0**

The

ratio

of u

Ca/

uCr(

mM

/nM

)

2 HALOvehicle Dex

14 HALOvehicle Dex

(a)

- 29 -

Fig. 6

0

20

40

60

80

100

120

140

Urin

ary

CTx

(ng/

mM

crea

tinin

e)

2 HALOvehicle Dex

14 HALOvehicle Dex

0

20

40

60

80

100

120

140

Urin

ary

CTx

(ng/

mM

crea

tinin

e)

2 HALOvehicle Dex

14 HALOvehicle Dex